The Complement System and its Activation
The C system consists of some 30 plasma and cell membrane proteins. Along with three other proteolytic cascades in blood (coagulation, fibrinolytic and kinin-kallikrein systems), it plays an essential role in maintaining life. In particular, the C system provides the first line of defence against microbial or other pathogenic attacks, actively participating in and also orchestrating their clearance, and, at the same time, augmenting the body's specific immune response. Although the C system is best known for its role in immunity, the system also plays a key role in conception, tissue regeneration and in many more physiological functions (1-3).
It is well known in the art that 1) C activation can proceed via three pathways (classical, alternative and lectin), each involving different C proteins; 2) C activation leads to the liberation of anaphylatoxin (C3a, C5a) which then activate mast cells, basophils, platelets, and other inflammatory cells with resultant liberation of inflammatory mediators (histamine, PAF, prostaglandins, etc.); 3) the latter “secondary” mediators set in motion a complex cascade of respiratory, hemodynamic and hematological changes, helping the body's self-defence, but also causing adverse effects. Among these, certain drug-induced hypersensitivity reactions (HSRs) have been shown to be associated with C activation and were therefore called C activation-related pseudoallergy (CARPA) (4, 5). CARPA is particularly important for this invention, as we use animal models of CARPA to provide examples for the invention.
It is furthermore well known from the literature that the production of C4b and C3b can label cells for phagocytic uptake by macrophages, a process known as opsonisation, and that the membrane attack complex (MAC), which forms in the final steps of the activation cascade, can cause direct lysis of invader cells, as well as activation of inflammatory cells. Altogether, C activation is a complex, multifaceted immune phenomenon that has been the subject of clinical and basic research for many decades (6-44).
The C3 Convertases
This invention is based on the phylogenetic conservedness of C3 convertase and, hence, substitutability of human C3 by animal C3 without major loss of function. The C3 convertase is a multiprotein complexes that assembles upon C activation, and converts C3 to C3a and C3b (FIG. 1). Its structure and function have been studied in great detail since the early seventies (7-44). It has two types, called classical pathway (CP) and alternative pathway (AP) C3 convertases. Both can form on the surfaces of pathogens or particles that the immune system recognizes as foreign. The CP C3 convertase is formed from membrane-bound C4b after its binding of the protease C2a, while the AP C3 convertase is formed from membrane-bound C3b upon its binding of the protease Bb. These C3 convertases have the same activity, catalyzing the deposition and than covalent binding of a large numbers of C3b molecules on the activator surfaces, which enhances the visibility and uptake of the particles by macrophages, a phenomenon called opsonisation. One important role of the AP of C activation is the amplification of CP C3 convertase function to yield substantially more C3b molecules on the pathogen (45). Surface bound C3b nucleates the formation of CP and AP C5 convertases, which convert C5 to C5a and C5b. The former is the most potent anaphylatoxin, while the latter nucleates the membrane attack complex (MAC, C5b-9), which has cytolytic activity. Part of the formed C5b-9 binds to S protein to form SC5b-9, a soluble form of MAC, one of the possible end products of the PS-C3 method of this invention.
Existing Assays for Complement Activation in Man and Animals
There are a large number of different assays measuring C activation in human or animal blood, plasma or serum (3, 6, 46-90). Some of these assays are based on quantitation of protein split products arising upon the limited proteolysis during C activation by ELISA, RIA (rocket) or electrophoresis (91). To measure C activation in humans, commercial kits are available from a few companies, including Quidel's (San Diego, Calif.) MicroVue series kits measuring human C3a, C5a, C4d, Bb, SC5b-9 and CH50 (92-95). Membrane-bound C byproducts can be measured by Western blot or other immune histological analysis. Yet other assays of C activation measure the functional activity of the C cascade using hemolysis as endpoint. These hemolytic assays, also known as CH50 assays, usually utilize sheep or rabbit red blood cells (SRBC, RRBC), and quantitate the consumption of multiple activable (precursor) C proteins along the C cascade. In this reaction the rate-limiting factor is the C protein whose concentration is the lowest in the sample, which can significantly vary among different species and samples. Because the endpoint of haemolytic assays is a biological reaction (hemolysis), these assays are difficult to standardize (63, 70, 86, 88, 91, 93, 95-102). The spectrum of animals where haemolytic assays can be used has not been fully explored.
As for the measurement of C activation in animals, human ELISAs can be used for such purpose, in cases when the human antibodies used in the ELISA cross-react with the corresponding animal antigen. Examples include the measurement of baboon and old world (synomolgus) monkey C3a, Bb, C4d and SC5b-9 by the respective human ELISAs from Quidel. ELISA kits for animal C split products are also available from a few vendors, that measure rat C3, C3a, C5a, guinea pig C3a and C5a, dog SC5b-9, Bb, C4d and mouse C3a. It is also possible to estimate C activation in animals in vivo by measuring physiological or blood chemistry changes that are known to be due, or at least associated with C activation. These measurements include the recording of cardiovascular and hemodynamic changes (e.g., rise or fall of systemic and pulmonary arterial pressure, heart rate), ECG changes, blood levels of thromboxane A2, etc. (105-122).
The Invention
The invention provides a common, universal assay for measuring C3 protein levels and consumption in animals by way of measuring the rise of human C split products using specific human ELISAs. Thus, human C split products serve as surrogate markers for animal C in blood, serum or plasma samples. In particular, the invention teaches the use of the human SC5b-9, CH50, C3a and C5a ELISA kits (such as marketed by Quidel Corporation, San Diego, Calif.) to measure the levels of C3 and C4 proteins in samples of different animals, whose level can be taken as measures of prior C activation. The invention utilizes a C3 specificity converting protein matrix or C4 specificity converting protein matrix for the determination of C3 and C4, respectively, which are mixed with the animal specimens, together with a known C activating substance, such as zymosan or HAGG. The mixture is incubated at a temperature between 36-38° C. for a sufficient period allowing development of human C3a, C5a and/or SC5b-9, which are measured with standard human ELISA or other human C5 and/or SC5b-9 measuring methods. The latter parameters indirectly quantitate the levels of C3 and/or C4 levels in the animal samples. Table 1 FIG. 1 shows the major steps involved in measuring animal C3 or C4 levels according to the invention.
The C3-SCM and C4-SCM allow the conversion of animal-specific C3 and C4 to be detectable and measurable by human-specific ELISAs. These matrices consist of C3 or C4-depleted normal human sera (C3depl-NHS, C4depl-NHS, marketed for example by Quidel Corporation, San Diego, Calif.), zymosan and/or HAGG or other C activator that trigger “intra-test activation”, which show the total activable amount of C3 and C4 in the animal samples. It is important to distinguish “intra-test C activation” from “pre-test C activation”, since the former is a necessary, unavoidable step in the assay, while “pre-test C activation” is a variable that may or may not be present, or may not be measurable by the PS-C3/C4 method of this invention. Also, because intra-test and pre-test C activation may utilize the same activator, e.g., zymosan or HAGG, it is easy to mix up or misinterpret the two processes.
The intra-test C activation involves incubation at preferably 37° C. of the test specimens of animal origin with C3-depleted (C3depl) or C4-depleted (C4depl) normal human serum (NHS) and measuring the production of human SC5b-9 (or C5a, or C3a) by ELISA following stimulation with zymosan or other C activator substance, such as HAGG. With animal C3 or C4 playing a rate-limiting role in the stimulated formation of human C activation by products, C3a, C5a or SC5b-9, the assay quantitates the level of animal C3 or C4. Because the actual levels of these proteins reflect previous C3 and/or C4 consumption, i.e., C activation, the assay enables quantitation of C activation in animal samples.
The test provides a quantitative measure of the level of active, i.e., activable C3 or C4 in the animal sample, taking the human SC5b-9 (or C3a, or C5a) as surrogate markers of animal C3 and C4. The total activable C3 or C4 in the sample is an ELISA OD in the 0-3.00 interval, obtained by subtracting the rise of C5a/SC5b-9 in the zymosan-free sample from that obtained in the zymosan-containing sample. The latter depends on the C status of animal samples, i.e., prior activation. Thus, the assay allows time-dependent quantitation of C activation in animal samples.
During intra-test C activation using animal-C3PM hybrid serum/plasma, the rate limiting step in the formation of human C5a or SC5b-9 is the activity of hybrid C3 convertase, consisting of animal C3 and animal and/or human C4b and C2a (classical pathway C3 convertase) or animal C3 and animal and/or human C3b and Bb (alternative pathway C3 convertase) (FIGS. 1A and B, respectively). The dark and white color circles in the figure illustrate the fact that the classical pathway hybrid C3 convertase builds up from animal C3 (dark) and animal (dark) and human (white) C4b and C2a, that are present in the complex in a ratio that corresponds to the volume ratio of animal to C3depl-NHS. Likewise, the alternative pathway C3 convertase builds up from animal C3 (dark) and animal (dark) and human Bb (white).
In C3depl-NHS, formation of human C5a or SC5b-9 will depend on the amount of animal C3 in the sample, which depends on prior C activation. Similar considerations apply to the C4depl-NHS assay, inasmuch as during the intra-test C activation using animal-C4 hybrid serum/plasma, the rate limiting step in the formation of human C5a or SC5b-9 is the activity of Clq2r2s, consisting of animal and/or human Clq, 2 animal and/or human Clr, and 2 animal and/or human C2s (C2 and C4 convertase). Consequently, in C4depl-NHS, formation of human C5a or SC5b-9 will depend on the amount of animal C4 in the sample, which depends on prior C activation via the classical pathway.
Novelty of the Invention
The invention addresses the problem that the methodical arsenal of measuring C activation in animals is much narrower than that available for humans. As mentioned above, there are commercially available ELISAs measuring C proteins in animals, these are offered only by a few vendors and are available for only a few proteins and few species. Most recently, a Chinese company was named as source of ELISA kits measuring SC5b-9 and Bb in beagle dogs (103), however, a follow-up paper questioned the reproducibility and validity of data (104). As an alternative approach, hemolytic assays have been used to measure C activation in different animals, but these assays are difficult to standardize (57), relatively insensitive and are considered irreproducible (87).
The present invention is based on the evolutionary conservedness of the C system, which fact is utilized in C research by the use of the SRBC to measure C consumption in animal serum specimens. In the latter, SRBC assay, it is the cytolitic function of the terminal C complex (MAC), formed from the animal C proteins, that is utilized for the measurement, namely, SRBC hemolysis. However, that hemolyis depends on the build-up of the TCC on the surface of SRBC, involving activation of the CP and the terminal chain. Hemolysis therefore will depend on the levels in the sample of all animal C proteins along the CP (C1-9), thus, the SRBC assay measures the composite activity of the CP in the animal serum sample. In contrast, because the assay mixture in the SC-C3 method in the present invention contains all human C proteins necessary for the detection of human SC5b-9 except C3, if the C3depl-NHS contains C5-9 is sufficient amounts, or in excess, the PS-C3 method measures ONLY the levels of animal C3 (or C4), i.e., it is possible to calibrate the system with known amounts of animal C3 (or C4) to make the method work like standard ELISAs equipped with standards for calibration. The present invention of specificity converter protein matrix allows therefore specific, pan-species measurement of animal C3 (or C4), which has not been possible to date with the SRBC or other assays.
Based on the above considerations, the present discovery that the animal C3 is used by the human C3 convertase to produce SC5b-9 that is recognized by Quidel's SC5b-9 kit as a human neoantigen, represents a serendipitous observation that could not be inferred directly from the existence and use of the SRBC assay to measure animal C. To our best knowledge, nor is there example in biology that a human enzyme complex would utilize as its functional part proteins from a variety of animals without major loss of activity. This “promiscuity” of the human C3 is not obvious in the C literature even for experts with substantial experience in the art.
Therefore, the present invention provides a method for species-independent measurement of complement (C) activation in animals comprising the steps of taking samples in the range of 3-100 microliter of anticoagulated blood, plasma or serum of an animal (specimen), mixing the specimen with a specificity converting protein matrix (SCM), mixing to the specimen/SCM mixture an activator of the C system (Act), incubating the specimen/SCM/Act mixture at a temperature between 36° C. to 38° C. for a time of 5-120 min and determining the production of one or more human proteins by ELISA or other analytical methods.
Claim 2 describes method according to claim 1, wherein in step a) the samples of blood, plasma or serum are taken from bovine, chicken, goat, guinea pig, horse, mouse, pig, rabbit, rat, sheep, turkey or dog.
Claim 3 describes a method according to claim 1 or 2, wherein in step b) the mixing of the specimen with a specificity converting protein matrix (SCM) is done at a low specimen/SCM ratio of 1-4:9-6.
Claim 4 describes a method according to claim 1, wherein SMC in step b) is either C3-depleted normal human serum (C3depl-NHS) or lyophilized C3depl-NHS (lyoC3depl-NHS) or C4-depleted normal human serum (C4depl-NHS) or lyophilized C4-depleted normal human serum (lyoC4depl-NHS).
Claim 5 describes a method according to claim 1, wherein SCM in step b) is supplemented with purified or recombinant human C5, preferably at a concentration that presents in the total test volume concentration of C5 in human blood.
Claim 6 describes a method according to claim 1, wherein SCM in step b) is supplemented with purified or recombinant human C5, C6, C7 and C9, preferably at individual concentrations which in the total test volume present their physiological concentration in human blood.
Claim 7 describes a method according to claim 1, wherein SCM in step b) is a mixture of purified or recombinant human C5, C6, C7 and C9, preferably at individual concentrations which in the total test volume correspond to their physiological concentration in human blood.
Claim 8 describes a method according to claim 1, wherein in step c) the activator of the C system (Act) is zymosan (in the 0.1-10 mg/mL range), or HAGG (in the 0.1-20 mg/mL range), or a liposomal drug like Ambisome (in the 0.01-100 mg phospholipid/mL range), or a surfactant like Cremophor EL or PS-80 (in the 0.01-100 mg/mL range).
Claim 9 describes a method according to claim 1, wherein in step d) the incubating of the specimen/SCM/Act mixture is done at 37° C. for 45-60 min with shaking.
Claim 10 describes a method according to claim 1, wherein in step e) human SC5b-9 ELISA is as endpoint for the quantification of C activation in different animal sera.
Claim 11 describes a method according to claim 1, wherein in step e) human C5a ELISA is used as endpoint for the quantification of C activation in different animal sera.
Claim 12 describes a method according to claim 1, wherein in step e) human C3a ELISA is used as endpoint for the quantification of C activation in different animal sera.
Claim 13 describes a method according to claim 1, wherein in step e) a human CH50 kit is as endpoint for the quantification of C activation in different animal sera, which kit detects human SC5b-9.
Claim 14 describes a tool for assessing the immune toxicity of drugs, drug candidates, biomaterials and all molecules and agents that may exert adverse effects via C activation after exposure to blood or other C-containing body fluids, which tool a) utilizes the steps specified in claim 1, b) utilizes animal specimens such as blood, plasma or serum, c) is applicable in all animals, d) utilizes a specificity converter protein matrix, such as specified in claims 4-7, e) utilizes ELISA kits specified in claims 10-13.